3 research outputs found
The structural topology of wild-type phospholamban in oriented lipid bilayers using 15N solid-state NMR spectroscopy
For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13° ± 6° with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant
Magic Angle Spinning NMR Reveals Sequence-Dependent Structural Plasticity, Dynamics, and the Spacer Peptide 1 Conformation in HIV‑1 Capsid Protein Assemblies
A key stage in HIV-1 maturation toward
an infectious virion requires
sequential proteolytic cleavage of the Gag polyprotein leading to
the formation of a conical capsid core that encloses the viral RNA
genome and a small complement of proteins. The final step of this
process involves severing the SP1 peptide from the CA-SP1 maturation
intermediate, which triggers the condensation of the CA protein into
the capsid shell. The details of the overall mechanism, including
the conformation of the SP1 peptide in CA-SP1, are still under intense
debate. In this report, we examine tubular assemblies of CA and the
CA-SP1 maturation intermediate using magic angle spinning (MAS) NMR
spectroscopy. At magnetic fields of 19.9 T and above, outstanding
quality 2D and 3D MAS NMR spectra were obtained for tubular CA and
CA-SP1 assemblies, permitting resonance assignments for subsequent
detailed structural characterization. Dipolar- and scalar-based correlation
experiments unequivocally indicate that SP1 peptide is in a random
coil conformation and mobile in the assembled CA-SP1. Analysis of
two CA protein sequence variants reveals that, unexpectedly, the conformations
of the SP1 tail, the functionally important CypA loop, and the loop
preceding helix 8 are modulated by residue variations at distal sites.
These findings provide support for the role of SP1 as a trigger of
the disassembly of the immature CA capsid for its subsequent <i>de novo</i> reassembly into mature cores and establish the importance
of sequence-dependent conformational plasticity in CA assembly